In order to fulfill its critical functions, eukaryotic topoisomerase I (topo I) transiently breaks DNA, forming a covalent linkage between the side chain of its active site tyrosine and a 3' phosphoryl of DNA, and subsequently reversing the covalent linkage to religate the DNA. Although this activity is important for survival, topo I can be toxic. As a result of natural or drug-induced accidents, the religation step can be hindered; topo I then stays on the DNA as a covalent "cleavable complex". Cells can apparently tolerate one or more cleavable complex but an encounter with a replication fork converts this structure into double-stranded break (DSB). Unless this is repaired, the cell will die.By definition, repair of damage associated with the covalent complex must involve the removal of the protein from the DNA. Although it can be imagined that removal would excise a chunk of DNA together with the associated protein, a more focused repair pathway was suggested by our discovery of an enzymatic activity that cleaves precisely a tyrosyl-phosophodiester (TP) bond located at the 3' end of DNA. We subsequently identified a gene (TDP1) that encodes a yeast enzyme with such activity. Homologs of yTDP1 are found in a wide variety of eukaryotic species and, since their amino acid sequences bear no obvious relation to any previously described protein, appear to constitute a novel enzyme family. Does TDP1 acts to prevent or to repair a topo I-associated DSB? On one hand, the enzyme might cleave topo I from a single strand break in duplex DNA. This would permit sealing of the break by DNA ligase and would thus avert creation of a DSB during passage of the replication fork. On the other hand, TDP1 could act only after a DSB occurs. We have addressed this question by examining the catalytic activity of yTDP1 on several different substrates that model these two stages. The experiments show that the enzyme works best on the kind of DNA structures found after a DSB has occurred. In the process, we discovered that the yeast AP endonuclease/3'-diesterase APN1 also can hydrolyze a TP bond from DNA. In contrast to TDP1, APN1 is most active on TP bonds presented in the middle of a DNA duplex. APN1 therefore has a substrate specificity complementary to TDP1, suggesting that it might act in the prevention of DSBs. We assessed the relative importance of TDP1 and APN1 in repair by in vivo assays on the relevant mutants after topo I damage. In previous work, the contribution of TDP1 to repair was only seen in a strain which had been genetically sensitized to reduce the efficacy of putative alternative repair pathways. We have now shown that TDP1 can make a significant difference to processing of topo I damage even without such sensitization. In contrast to the substantial changes in the capacity of tdp1 mutants to handle the cleavable complex and its derivatives, under all circumstances that we tested, apn1 mutants were indistinguishable from their wild-type counterparts. We conclude that topo I damage is predominantly fixed by DSB repair and that this pathway has a strong dependence on the capacity of TDP1 to clean the ends of the break so as to permit subsequent steps.